The DNA intercalator doxorubicin is a highly effective chemotherapeutic agent used to treat several cancer types including breast cancer The mechanism of toxicity of DNA intercalators is generally attributed to their ability to inhibit topoisomerase II which results in the generation of double strand breaks in DNA. Emerging studies report that DNA intercalators are capable of modulating epigenetic modifications, specifically inhibiting DNA methylation, resulting in reexpression of silenced genes. These studies were supported in this laboratory with both doxorubicin and mitoxantrone. Previous studies in this laboratory have discovered a novel function for doxorubicin as it is capable of inhibiting the activity of the DNA methyltransferase DNMT1 in vitro. These studies also determined that DNMT1 is an important contributor to the apoptotic activity of doxorubicin. The objectives of this proposal are to characterize the mechanism of intercalator mediated epigenetic alterations and how DNA methyltransferase inhibition contributes to the cytotoxic effects of these drugs. Improved understanding of the the molecular basis of DNA intercalator mediated cytotoxicity will allow for more effective use of the drugs clinically. The specific aims are (1) to characterize the effects of DNA intercalators on DNA methyltransferase activity and interaction with DNA in vitro and (2) to characterize the effects of DNA intercalators on DNA methylation patterns and histone modifications in vivo. The DNMT-magnetic Beads Assay, which was previously developed in this laboratory, will be used to analyze the effects of DNA intercalators on methyltransferase activity using recombinant DNMT1, DnmtSa, and DnmtSb. Gel shift assays will be used to monitor the effects on enzyme and DNA binding interactions in vitro. In order to monitor the effects of DNA intercalators on DNA methylation in vivo, the methylation status of silenced genes in DNA intercalator treated cells will be measured both qualitatively using methylation specific PCR and quantitatively with bisulfite genomic sequencing. The effects, on gene expression will be determined using real-time RT-PCR. Global methylation will be measured using HPLC and changes in histone modifications will be studied using chromatin immunoprecipitation. Public Health Relevance: The results of this study will improve the understanding of how an important and commonly used class of chemotherapeutic drugs like doxorubicin is capable of killing cancer cells. This knowledge will enable them to be used more effectively in the clinic, which is expected to improve the outcome for cancer patients.